Resilience to aging, stress tests, biomarkers, senescence, age-related disease, enhancing resilience, animal models of resilience

The 2019 Barshop Conference on Aging was held at the Mayan Dude Ranch in Bandera, TX with the theme “Resilience in Aging and Age-related Disease”. The conference was sponsored by the Barshop Institute for Longevity and Aging Studies at the University of Texas Health San Antonio, San Antonio, TX, and organized by Sara Espinoza, MD, MSc, Adam Salmon, PhD, and Amrita Kamut, PhD. Presentations consisted of platform sessions and posters.
The first platform session presented an Overview of Resilience: Defining and Predicting. The speakers were Dr. Francesca Maccharini, National Institute on Aging (NIA’s view on resilience), Dr. Jeremy Watson, Johns Hopkins University School of Medicine (Physical frailty in older adults: Causes and interventions), and Dr. Cathleen ColonEmeric, Duke University School of Medicine (Predicting physical resilience in older adults).
The second session was on Identifying Biomarkers for Resilience. The speakers were Dr. Derek Huffman, Albert Einstein College of Medicine (Developing dynamic assays to quantify resilience in mice), Dr. Mini Jacob, University of Texas Health San Antonio (Gait: A biomarker of resilience), and Dr. Jamie Justice, Wake Forest University School of Medicine (Biomarkers of aging for geroscienceguided clinical trials).
The third session was on Cell Senescence in Resilience. The speakers were Dr. James Kirkland, Mayo Clinic (Aging, senolytics, age-related disease, and resilience), Dr. Mitzi Gonzales, University of Texas Health San Antonio (Pilot investigation of senolytic therapy in Alzheimer’s disease), and Dr. Nathan LeBrasseur, Mayo Clinic (Ageassociated changes in measures of physical resilience in mice).
The fourth session was on Intervention for Resilience. Can Resilience be Modified? The speakers were Dr. George Kuchel, University of Connecticut Center on Aging (Immune responses to vaccination in aging: Translational insights into immune resilience), Dr. Monica Serra, University of Texas Health San Antonio (Adjusting after stroke: Lessons to promote resilience during recovery), Dr. James White, Duke University School of Medicine (Chronic exposure to youthful circulation improves resilience in old mice), Dr. Alessandra Bartolomucci, University of Minnesota (Social stress drives senescence), and Dr. Arlan Richardson, University of Oklahoma Health Science Center (The effect of age and dietary restriction on resilience in mice).
The fifth and final session was on Big Data Biology to Address Resilience. The speakers were Dr. Warren Ladiges, University of Washington School of Medicine (Physical resilience is a predictor of healthy aging in mice), Dr. Ravi Varadhan, Johns Hopkins University School of Medicine (Dynamical systems thinking for studying resilience to clinical stressors), Dr. Sudha Seshadri, University of Texas Health San Antonio (Biology of dementias: Insights from fenetics, omics, and systems biology approaches), and Dr. Jamie Walker, University of Texas Health San Antonio (Primary age-related tauopathy (PART) and successful aging: Resistance and resilience to neuropathologic changes and cognitive decline in aging).

The critical endoplasmic reticulum (ER)-associated protein quality control (ERQC) adaptation of the longlived Caenorhabditis elegans rpn-10(ok1865) proteasomal mutant

Meghna N. Chinchankar^a,b,c*, Karl A. Rodriguez^a,d, Alfred L. Fisher^a,b,c,e

^a Barshop Institute for Longevity and Aging Studies, UT Health Science Center at San Antonio (UTHSCSA), San Antonio, TX. ^b Center for Healthy Aging, School of Medicine, UTHSCSA, San Antonio, TX. ^c Division of Geriatrics, Gerontology, and Palliative Medicine, Department of Medicine, UTHSCSA, San Antonio, TX. ^d Department of Cell Systems and Anatomy, UTHSCSA, San Antonio, TX. ^e Division of Geriatrics, Gerontology, and Palliative Medicine, Department of Medicine, University of Nebraska Medical Center, Omaha, NE.

Protein degradation mechanisms are integral for preserving the proteome. Their reduced efficiency leads to protein aggregation which potentiate several proteotoxic disorders. Paradoxically, we reported that the Caenorhabditis elegans rpn-10(ok1865) proteasome mutant exhibits enhanced proteostasis, elevated stress resistance and extended lifespan. RPN-10/PSMD4 is a ubiquitin receptor on the 26S proteasome that targets poly-ubiquitinated substrates to its catalytic core for degradation. Moreover, proteasome dysfunction of the rpn-10 mutant is characterized by reduced, not inhibited, ubiquitin fusion degradation. To elucidate the suite of protective mechanisms, we found that the ER membrane and ER-associated degradation (ERAD) genes were significantly upregulated in the rpn-10 mutant. Thus, we hypothesized that the rpn-10 mutant exhibits enhanced ERQC. Accordingly, the rpn- 10 mutant exhibits higher ER stress resistance and altered ER homeostasis. Complementarily, attenuated expression of the aggregation-prone α-1 antitrypsin (ATZ) reporter proves that ER proteostasis is also ameliorated in the rpn- 10 mutant. Via a forward genetics screen for suppressors of decreased ATZ aggregation in the rpn-10 mutant, we identified a novel player, H04D03.3, which is a putative homolog of the proteasome-associated adaptor protein ECM29. This finding unexpectedly suggests that the assembly of the rpn-10 mutant proteasome distinctly regulates its ERQC. Further, we observed that cytosolic proteostasis and longevity benefits are contingent on the ER master chaperone hsp-3/-4 (BiP) and ER ATPase cdc-48.2 (p97/VCP), thus highlighting the significance of ERQC in the rpn-10 mutant. Altogether, it appears that mild proteasomal dysfunction induces a unique ERQC adaptation that underlies improved proteostasis and increased longevity of the rpn-10 mutant.

Correspondence to: Meghna N. Chinchankar, Barshop Institute for Longevity and Aging Studies, UT Health Science Center at San Antonio (UTHSCSA), San Antonio, TX.
Email: chinchankar@uthscsa.edu

The protein translation paradox and its implications for the onset of proteostasis collapse and aging

Harper Kim^a,b,c*, Andrew Pickering^b,c

^a South Texas Medical Scientist Training Program (STX-MSTP), UT Health San Antonio. ^b Department of Molecular Medicine, UT Health San Antonio. ^c Sam & Ann Barshop Institute for Longevity and Aging Studies.

High levels of protein translation have been shown to cause age-related functional declines. However, protein translation is high only in young adulthood. Protein translation sharply declines thereafter, staying at a very low level at mid-old ages in many organisms, including humans. These studies collectively raise the protein translation paradox: High protein translation causes agerelated functional declines, but protein translation declines pronouncedly with age. To resolve these contradictory findings, we hypothesize that a rise in protein translation during early adulthood initiates aging at the molecular level by overwhelming protein quality control and triggering disruption of protein homeostasis (proteostasis); consequently, protein translation is repressed with age as an adaptive response to improve proteostasis, slow down aging, and optimize the lifespan. Consistent with the hypothesis, our data suggests that long-lived fly strains (e.g. chico homozygotes) with no signs of early proteostasis collapse, unlike wild types, do not enhance protein translation in early adulthood. To directly test our hypothesis, we transiently modified expression levels of translational machineries in early, late, or whole adulthood of fruit flies via the GeneSwitch (drug-inducible) system. This approach enables us to 1) prevent/enhance a rise in protein translation during early adulthood and 2) block agedependent decline in protein translation. We will examine how these affect life- and health span and levels of polyubiquitinated protein aggregates. Additionally, we will induce early life increase in protein translation in long-lived fly strains and examine whether this abolishes their longlived phenotypes.

Correspondence to: Harper Kim, South Texas Medical Scientist Training Program (STX-MSTP), UT Health San Antonio.
Email: kimsw@livemail.uthscsa.edu

Chronic subordination stress induces tissue-specific cellular senescence in mice

Carey Lyons^a,b*, Maria Razzoli^a, Rachel Mansk^a, Giada Caviola^a,c, Kohinoor Kahn^a, Madeleine Berg^a, Judith Campisi^d,e, Jan Van Deursen^f, Alessandro Bartolomucci^a

^a Department of Integrative Physiology and Biology . ^b Graduate Program in Neuroscience University of Minnesota, Minneapolis Minnesota. ^c Universitá di Parma, Parma Italy. ^d Buck Institute for Research on Aging, Novato CA. ^e Lawrence Berkeley National Laboratory, Life Sciences Division, Berkeley CA. ^f Dept. Biochemistry and Molecular Biology, Mayo Clinic, Rochester MN.

Conventional wisdom posits that severe stress ages a person. Scientific evidence backs this claim, showing that higher perceived stress levels correlate with shorter telomeres, a trigger for cellular senescence. Senescent cells (SNCs) accumulate with age and contribute to the onset and progression of numerous aging-related diseases. Our lab previously showed that lifelong chronic subordination stress (CSS) in mice shortens lifespan and increases senescence markers. In this study, we asked whether briefer exposure to mild (restraint) or severe (CSS) stress would be sufficient to increase accumulation of SNCs in young or old mice. We found that in young mice, 4 weeks of CSS, but not restraint, increased expression of senescence markers in a tissue-dependent manner with changes particularly evident in the brain and adipose. Targeted clearance of p16-positive cells in p16-3MR mice normalized expression of senescence and SASP markers, causally linking stress with SNCs accumulation. We also found that while, as expected, natural aging increases expression of senescence markers in the brain, 4 weeks of CSS in old mice did not further augment expression. These results are the first to demonstrate that psychological stress increases abundance of SNCs, implicating them as mediators of stress-induced disease. Our findings mark an important advance toward identifying stress-associated mediators responsible for SNCs accumulation and their impact on aging-related diseases.

Correspondence to: Carey Lyons, Department of Integrative Physiology and Biology, Graduate Program in Neuroscience University of Minnesota, Minneapolis Minnesota.
Email: lyons293@umn.com

Effects of chronic exposure to aluminum ions on cellular resilience

Kennedy S. Mdaki^a* , Jonathan Dorigatti^a, Kevin Thyne^a, Yuhong Liu^a, Adam Salmon^a

^a Sam and Ann Barshop Institute for Longevity and Aging Studies, UT Health at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245.

Introduction. Aluminum (Al) is one of the most abun￾dant metals on earth’s crust and chronic exposure leads to measurable accumulation in humans. Al ions (Al³⁺) are a significant cause of oxidative stress generated by mitochondria. Senescent cells are known to have altered mitochondrial bioenergetics, generate oxidative stress, and contribute to chronic inflammation in humans. Accu￾mulation of senescent cells is a significant driving process of aging and aging-related diseases. Aim. We investigated whether chronic exposure to Al³⁺ promotes the develop￾ment of cellular senescence. Methods. Using primary fi￾broblast cells from the skin of young mice, cellular senes￾cence was determined by analyzing the effects of chronic exposure to graded doses of aluminum chloride (AlCl₃) on cell proliferation, viability, and expression of senescence associated beta-galactosidase(SAβ-gal) using an IncuCyte S3 live cell analysis system. Mitochondrial dynamics were assessed using confocal live cell imaging and west￾ern blot analysis. Cellular bioenergetics were assessed using a seahorse XF24. Results. With increasing length of AlCl₃ exposure, we found decreased cellular proliferation, increased senescence associated β-gal, and altered bio￾energetics. Expression of p53 (a regulator of senescence) and DRP1 (a regulator of mitochondrial fission) were elevated in the AlCl₃ treated cells. Discussion. Our work provides vital initial insights onto the potential role of Al³⁺ on cellular senescence and possible implications on the progression of mammalian aging and disease. Future work will expand the scope of this work by determining the expression of additional markers of senescence, oxidative damage, and mitochondrial dynamics in response to chronic exposure to Al³⁺ in mammals.

Correspondence to: Kennedy S. Mdaki, Sam and Ann Barshop Institute for Longevity and Aging Studies, UT Health at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245.
Email: mdaki@uthscsa.edu

Tau-induced endothelial cell impairment as a driver of microvascular dysfunction in Alzheimer’s disease

Andy Banh^a,b*, Stacy Hussong^a,b, Candice Van Skike^a,b, Angela Olson^a,b, Stephen Hernandez^a,b, Rakez Kayed^c , Veronica Galvan^a,b

a^a University of Texas Health Science Center at San Antonio. ^b Barshop Institute for Longevity and Aging Studies. c University of Texas Medical Branch.

Cerebrovascular dysfunction is the first and most abnormal biomarker in Alzheimer’s disease (AD) progression. We showed that pathogenic soluble tau aggregates, which are causally implicated in AD and spread transneuronally, also accumulate in cerebrovascular endothelial cells where they may play a role in AD cerebrovascular dysfunction. Indeed, we found that pathogenic tau impairs endothelium-dependent cerebral blood flow (CBF) responses in P301S(PS19) mice modeling tauopathy. Similar to neuron-to-neuron transmission, soluble tau aggregates were transmitted to human brain microvascular endothelial cells (HBEC), where they decreased microtubule stability and endothelial nitric oxide synthase (eNOS) activation and induced senescence as cycle arrest and the development of a senescence associated secretory phenotype (SASP). Consistent with these observations, microtubule destabilization, eNOS deactivation, and senescence/ SASP were significantly increased in microvasculature of P301S(PS19) mice modeling tauopathy. The mechanisms by which pathogenic tau contributes to cerebrovascular dysfunction in AD, however, are not defined. To answer this question, we measured endothelium-dependent vasodilation in P301S(PS19) mice in which soluble tau aggregates were removed with immunotherapy or remained with control treatment. Removal of soluble tau aggregates was sufficient to restore endothelium-dependent CBF in P301S(PS19) mice. These data indicate that soluble tau aggregates mediate deficits in endotheliumdependent CBF responses in a mouse model of tauopathy and which may be caused by impaired eNOS activation and induction of endothelial cell senescence/SASP. Thus, our studies indicate that soluble tau aggregates mediate cerebrovascular dysfunction in a model of tauopathy and suggest that pathogenic tau removal may hold promise for the treatment of AD and other tauopathies.

Correspondence to: Andy Banh, University of Texas Health Science Center at San Antonio.
Email: banh@livemail.uthscsa.edu

Tau-induced astrocyte senescence: A novel mechanism for neural dysfunction in Alzheimer’s disease.

Angela Olson^a,b*, Stacy Hussong^a,b,c, Aubrey Sills^b, Rakez Kayed^d, Veronica Galvan^a,b,c

^a Department of Cellular & Integrative Physiology, University of Texas Health at San Antonio, TX, USA. ^b Barshop Institute for Longevity and Aging Studies, University of Texas Health at San Antonio, TX, USA. ^c South Texas Veterans Heath Care System, San Antonio, TX, USA. ^d Department of Neurology, University of Texas Medical Branch at Galveston, TX USA.

Chronic sterile inflammation is a pathological feature of Alzheimer’s disease (AD) and other neurodegenerative diseases. The mechanisms that drive neuroinflammation and its impact on AD progression are still incompletely understood. The accumulation of molecular damage in somatic cells can trigger cellular senescence, an irreversible state of cell cycle arrest accompanied by the expression of proinflammatory mediators known collectively as the “senescence-associated secretory phenotype” (SASP). Misfolded tau forms pathogenic soluble aggregates that are released extracellularly and are transmitted trans-neuronally, promoting native tau phosphorylation and aggregation in target cells. We recently showed that, in addition to neurons, pathogenic soluble extracellular tau aggregates propagate to brain microvascular endothelial cells, where microtubule destabilization triggers senescence/SASP. Because astrocytes have a critical role in the regulation of both synaptic function and cerebral blood flow and are directly exposed to tau at its site of release at the tripartite synapse, we conducted studies to define whether soluble aggregated tau propagates to astrocytes, inducing astrocyte senescence/SASP and neuronal dysfunction/damage. Our studies indicate that tau can be propagated transcellularly to astrocytes, triggering cellular senescence/SASP. Our studies suggest that astrocyte senescence is detrimental to dendritic and synaptic structure and density, suggesting that pathogenic soluble tau-induced astrocyte senescence may contribute to synaptic dysfunction and loss in AD. Drugs that eliminate senescent cells are FDA-approved and antibody-based approaches to remove tau from brain are already in clinical trials. Our studies suggest that these interventions could be effective in the treatment of AD and other tauopathies.

Correspondence to: Angela Olson, Department of Cellular & Integrative Physiology, University of Texas Health at San Antonio, TX, USA.
Email: olsona@livemail.uthscsa.edu

Oligomeric tau contributes to cerebrovascular deficits through nitric oxide synthase dysfunction

Candice E. Van Skike, Ph.D.^a*, Andy Banh, B.S.^a , Stacy A. Hussong, Ph.D.^a, Veronica Galvan, Ph.D.^a

^a University of Texas Health San Antonio, San Antonio, TX.

Brain microvascular deficits were recently identified as the earliest and most abnormal biomarker in AD progression. We recently reported the accumulation of pathogenic soluble tau aggregates (oligomers) in cerebromicrovasculature of AD and other tauopathies. The impact of cerebrovascular accumulation of pathogenic tau, however, is not yet understood. To define the functional consequences of cerebrovascular tau we used laser Doppler flowmetry to assess microvascular function as (a) the coupling of cerebral blood flow (CBF) to neuronal activity (neurovascular coupling, NVC) and (b) endothelium-dependent vasodilation in mice modeling tauopathy where pathogenic tau was removed with immunotherapy, or remained in brain. Changes in CBF in barrel cortex as a result of whisker stimulation or topical acetylcholine application provided measures of NVC and endothelium-dependent vascular reactivity respectively. The contributions of neuronal (nNOS) and endothelial (eNOS) nitric oxide synthases to NVC were determined through the use of nNOS-selective and general NOS inhibitors. Our data show that isolated overexpression of mutant human tau in the P301S(PS19) model of tauopathy is sufficient to impair barrel cortex NVC, and that this deficit is mediated by a profound reduction of nNOS activity. Tauopathy in P301S(PS19) mice also impaired eNOS-dependent cortical vasoreactivity, a deficiency that was mitigated by removal of pathogenic tau aggregates with immunotherapy. Our data suggest that the inhibition of NOS activation by pathogenic soluble tau aggregates may underlie microvascular dysfunction in tauopathies, including AD. Therapeutic removal of pathogenic tau may be used to delay disease onset and/or slow progression of AD and potentially other tauopathies.

Correspondence to: Candice E. Van Skike, University of Texas Health San Antonio, San Antonio, TX.
Email: vanskike@uthscsa.edu

Deficits in nonsense-mediated RNA decay contribute to increased translation and neuronal death in tauopathy

Gabrielle Zuniga^a*, Bess Frost^a

^a University of Texas Health San Antonio, Cell Systems and Anatomy, San Antonio, TX.

Cells utilize the nonsense mediated RNA decay (NMD) pathway for post-transcriptional control of gene expression and degradation of aberrant RNA that could otherwise encode potentially toxic proteins. We have recently reported that transgenic expression of pathogenic tau, a protein that accumulates in Alzheimer’s disease and related “tauopathies,” promotes neurodegeneration by increased nuclear export of polyadenylated RNA through nuclear envelope invaginations in Drosophila and human tauopathy. Nuclear pores line these invaginations and facilitate rapid nuclear export of polyadenylated RNA in Drosophila and human tauopathy. Increased mRNA export may lead to increased cytoplasmic RNA that overwhelms the ability of NMD factors to degrade aberrant or error-containing RNA. We find that genetic manipulation of NMD machinery, but not other RNA quality control pathways, significantly modifies tau-induced neurotoxicity. In addition, the ability of NMD to clear aberrant RNA transcripts was shown to decrease with age and increase after genetic manipulation of RNA export factors in control and tau transgenic Drosophila. These results are consistent with the finding that global translation is increased in adult tau transgenic Drosophila and Drosophila with RNAi-mediated knockdown of core NMD factors. Our preliminary studies suggest a necessary role of NMD in post-transcriptional control of gene expression by preventing accumulation of aberrant RNA transcripts and translation into proteins that are toxic to neurons in tauopathy.

Correspondence to: Gabrielle Zuniga, University of Texas Health San Antonio, Cell Systems and Anatomy, San Antonio, TX.
Email: zunigag3@livemail.uthscsa.edu

Deficiency of a novel depalmitoylase exacerbates dietinduced obesity, insulin resistance, and inflammation

Jason Hadley B.S.^a*, Jiyoon Ryu Ph.D.^a, Lily Dong Ph.D.^a

^a University of Texas Health San Antonio, San Antonio, TX.

Protein palmitoylation is a reversible post-translational modification that has gained rapid attention for its ability to modulate a diverse range of signaling pathways in cells. Depalmitoylases are a class of enzymes that catalyze the removal of this lipid group from target proteins, which subsequently alters the subcellular localization of their target molecules. Recently, we identified LF8 as a potential novel depalmitoylase and found its transcriptional levels are positively associated with insulin sensitivity in mouse liver. Since LF8 is uncharacterized in mammals, we have generated LF8 whole body KO mice to test the physiological function. Deletion of the LF8 gene in mice leads to an exacerbated high fat diet-induced obesity phenotype and pronounced hepatic inflammation. Interestingly, we found LF8 expression to be largely restricted to peritoneal macrophages and its loss results in aberrant cytokine signaling. These results not only reveal an important role of depalmitoylation in regulating insulin sensitivity and diet-induced chronic inflammation, but also suggest that regulation of peritoneal macrophage function contributes to systematic energy homeostasis.

Correspondence to: Jason Hadley B.S., University of Texas Health San Antonio, San Antonio, TX.
Email: hadleyj@uthscsa.edu

Loss of vacuolar acidity results in iron sulfur cluster defects and divergent homeostatic responses during aging in Saccharomyces cerevisiae

Kenneth L. Chen^a, Kathleen L. Helge^b, Matt Kaeberlein^a, Brian M. Wasko^b*

^a Department of Pathology, University of Washington, Seattle, WA, USA 98195. ^a Department of Biology and Biotechnology, University of HoustonClear Lake, Houston, TX USA 77058.

The loss of vacuolar/lysosomal acidity is an early event during aging that has been linked to mitochondrial dysfunction. However, it is unclear how loss of vacuolar acidity results in age-related dysfunction. Through unbiased genetic screens, we determined that increased iron uptake can suppress the mitochondrial respiratory deficiency phenotype of yeast vma mutants, which have lost vacuolar acidity due to genetic disruption of the vacuolar ATPase proton pump. Yeast vma mutants exhibited nuclear localization of Aft1, which turns on the iron regulon in response to iron sulfur cluster (ISC) deficiency. This led us to find that loss of vacuolar acidity with age in wildtype yeast causes ISC defects and a DNA damage response. Using microfluidics to investigate aging at the single cell level, we observe grossly divergent trajectories of iron homeostasis within an isogenic and environmentally homogeneous population. One subpopulation of cells fails to mount the expected compensatory iron regulon gene expression program and suffers progressively severe ISC deficiency with little to no activation of the iron regulon. In contrast, other cells show robust iron regulon activity with limited ISC deficiency, which allows extended passage and survival through a period of genomic instability during aging. These divergent trajectories suggest that iron regulation and ISC homeostasis represent a possible target for aging interventions.

Correspondence to: Brian M. Wasko, Department of Biology and Biotechnology, University of Houston-Clear Lake, Houston, TX USA 77058.
Email: wasko@uhcl.edu

The aging immune system drives senescence and dysfunction of peripheral tissues

Matthew J. Yousefzadeh^a,b,c, Rafael R. Flores^a,b,c, Yuxiang Cui^d , Robert W. Brooks^a , Jing Zhao^a , Luise A. Angelini^a,b,c, Erin A. Wade^a , Christy E. Trussoni^e , Jonathan I. Kato^a , Collin A. McGuckian^a,b,c, Dong Wang^f , Qing Dong^f , Prashanti Patil^f , Ken Li^f , Aiping Lu^g,h, Ryan D. O’Kelly^a,b,c, Warren C. Ladigesⁱ , Christin E. Burd^j , Nicholas F. LaRusso^e , Smitha P.S. Pillai^k , Nam V. Vo^f , Eric E. Kelley^l , Yinsheng Wang^d , Johnny Huard^g,h, Paul D. Robbins^a,b,c*, Laura J. Niedernhofer^a,b,c*

^a Institute on the Biology of Aging and Metabolism, University of Minnesota. ^b Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota. ^c Department of Molecular Medicine and the Center on Aging, The Scripps Research Institute. ^d Department of Chemistry, University of California Riverside. ^e Division of Gastroenterology and Center for Cell Signaling in Gastroenterology. ^f Department of Orthopedic Surgery, University of Pittsburgh. ^g Steadman Philippon Research Institute. ^h Department of Orthopedic Surgery, McGovern Medical School, University of Texas Health Science Center at Houston. ⁱ Department of Comparative Medicine, University of Washington. ^j Departments of Molecular Genetics and Cancer Biology and Genetics, The Ohio State University. ^k Fred Hutchinson Cancer Research Center, Seattle. ^l Department of Physiology & Pharmacology, West Virginia University.

Aging of the immune system, or immunosenescence, prevents the elderly from mounting a productive immune response against pathogens or to vaccinations, contributing to infections that are common causes of death in the elderly. To define the contribution of immune aging to organism aging, Ercc1, which encodes a subunit of the DNA repair endonuclease ERCC1-XPF, was deleted only in hematopoietic cells of the mouse to increase endogenous DNA damage and oxidative stress. VaviCre+/-;Ercc1-/fl mice were healthy into adulthood, but then displayed both attrition and senescence of immune cell populations. The mice had accelerated, progressive onset of immunosenescence with reduced T, B and NK cell function. Remarkably, non-lymphoid organs also had increased senescence and oxidative stress, resulting in premature aging of liver, kidney and intervertebral disc and reduced repair capacity of injured muscle, indicating that senescent, aged immune cells contribute to systemic aging. Indeed, transplantation of splenocytes from Vav-iCre+/-;Ercc1-/fl or old wildtype mice induced senescence in multiple tissues in trans. Conversely, transplantation of immune cells from young mice into aged animals reduced the senescent cell burden in multiple tissues. Short-term treatment of VaviCre+/- ;Ercc1-/fl mice with rapamycin improved T and B cell function, demonstrating that immunosenescence is malleable. Taken together, these data demonstrate that an aged immune system contributes to driving systemic aging and represents a key therapeutic target to extend healthy aging.

Correspondence to: Matthew J. Yousefzadeh, Institute on the Biology of Aging and Metabolism, University of Minnesota.
Email: myousefz@umn.edu

Funding: This work was supported by the NIH grants P01 AG043376, U19 AG056278, R56 AG059676, K26 AG001057 and R01 AG044376, Glenn Foundation and the Irene Diamond/AFAR.

Identification of a novel adipokine tetranectin and its metabolic function

George Plasko^a*, Sija He^a , Jingjing Zhang^b , Fen Liu^b , Juli Baia^a,b, Lily Dong^c , Feng Liu^a,b

^a Department of Pharmacology, University of Texas Health at San Antonio, San Antonio, TX 78229. ^b Department of Metabolism and Endocrinology, Metabolic Syndrome Research Center, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China. ^c Department of Cell Systems & Anatomy, University of Texas Health at San Antonio, San Antonio, TX 78229.

Obesity has an established association with insulin resistance, type 2 diabetes mellitus, dyslipidemia, and atherosclerosis, with a heavy burden on the healthcare system. Preventing the progression from obesity to insulin resistance requires an understanding of the regulatory mechanisms involved in the loss of insulin sensitivity. Adipose tissue is well known to function as an endocrine organ that produces many kinds of adipokines, such as leptin, adiponectin, resistin, and retinol-binding protein 4 etc. In the current study, we report the identification and initial characterization of a novel adipokine tetranectin, the C-type lectin domain family 3 member B (CLEC3B) gene coded protein that is highly enriched in white adipose tissue. We found that the serum level of tetranectin was much higher in both obese and diabetic patients. Knocking out the tetranectin gene in mice protected against glucose intolerance in males but reduced insulin and glucose tolerance in females, without effects on food intake and body weight for either sex. Mechanistically, tetranectin targets liver tissues and its deficiency improves insulin-stimulated AKT phosphorylation at serine 473. Taken together, we have identified a novel adipokine which mediates metabolic crosstalk among tissues to maintain systemic glucose me tabolism. Further investigation of tetranectin’s function could yield a new target for the development of effective therapeutic treatment for obesity and its associated metabolic diseases.

Correspondence: George Plasko, Department of Pharmacology, University of Texas Health at San Antonio, San Antonio, TX 78229.
Email: plasko@livemail.uthscsa.edu

Monkeying with resilience: The marmoset as a model for pre-clinical intervention

Aubrey Sills^a , Joselyn Artavia^a , Adam Salmon^a*

^a Barshop Institute, UT Health San Antonio.

The challenge to recover from physiological issues that were once relatively easy to do in young age becomes increasingly more difficult with age. Effective models of resilience will improve the translation of interventions that address resilience in clinical application. While common animal models (e.g., rodents) have advantages in preclinical development, testing in nonhuman primate models may offer advantages as pre-clinical models of translation due to greater similarities in basic physiological functions with humans. Common marmosets are small, New World monkeys with a relatively short lifespan and small size compared to other primates. We have utilized marmosets as a pre-clinical nonhuman primate model of interventional geroscience as well to develop, through reverse translation, useful models of human aging pathology. We are identifying several potential markers of resilience in aging marmosets that might have clinical relevance in terms of interventions. Guided by established rodent and human frailty indices, we have developed a marmoset accumulation of deficits index that includes objective (body weight, medical records, etc.) and subjective assessments of functional deficits. In addition, we find specific hematological markers in aging marmosets that may be used to define overall resilience within the context of clinical relevance. Lastly, we are developing cellular resilience as a means to define biological resilience at an individual level using fibroblast cell lines derived from the skin of aging animals. Overall, defining the effect of age on resilience in the marmoset may aid in developing geroscience-based approaches to clinical intervention.

Correspondence to: Adam Salmon, Barshop Institute, UT Health San Antonio.
Email: salmona@uthscsa.edu

Left ventricular remodeling proceeds from young adulthood into midlife in intrauterine growth restricted baboons

Clarke GD^a*, Huber H^b , Li C^b , Kuo AH^c , Nathanielsz PW<sup>b

a</sup> UT Health Science Center San Antonio. ^b University of Wyoming.^c Massachusetts General Hospital.

Previous cross-sectional studies have shown young adult baboons (~5-6 y.o.) subjected to intrauterine growth restriction (IUGR), by maternal calorie restriction during pregnancy and lactation, exhibit ventricular remodeling with mildly impaired heart function relative to age/sex matched controls (CTL). The present study was designed to assess the degree of progression of this condition. METHODS: In this longitudinal study, cardiac MRI was performed on male IUGR baboons (n=7). A 3 Tesla, Siemens MRI system was used with phased array coils, parallel imaging acquisition and breath hold during the scan. Studies of IUGR animals occurred at 4.7+0.1 yr intervals; the first scan was at 5.8+1.2 y (human equivalent~24 years) and the second (scan 2) at 10.4+1.2 yr (human equivalent~40 years). Scan on the CTL animals (N=4) occurred at 5.3+1.4 years. RESULTS: Change in body weight over 4.7 years was less in the IUGR group (∆wt=6.3+6.1 kg) than in the CTL group (∆wt=11.5+8.2 kg). Left ventricular (LV) ejection fraction (EF) was significantly greater in IUGR animals in scan 2 (+10.7%, p=0.03) but not in normal controls (+1.8%). Stroke volume and end-diastolic LV volume were normalized to body surface area (BSA). SV/BSA (17.6+4.9, 31.5+12.3 mL m-2; p=0.016) and EDV/BSA (47.3+13.6, 64.5+18.8 mL m-2; p=0.045) also were significantly increased in IUGR animals but not in controls. In IUGR subjects, ∆wt was significantly and positively correlated with ∆EF (r=0.86. p=0.01). CONCLUSIONS: In IUGR, but not in CTL baboons, cardiac function adaptations continue into midlife and are related to increases in body weight with aging. We conclude that IUGR programs cardiovascular function and that programmed change continue into midlife.

Correspondence to: Clarke GD, UT Health Science Center San Antonio.
Email: clarkeg@uthscsa.edu

The impact of aging on vaccine-induced memory recall responses

Julia M. Scordo^a*, Tucker J. Piergallini^a , Colwyn Headley^a , Ron E. Banks^b , James F. Papin^b , Joanne Turner<sup>a

a</sup> Texas Biomedical Research Institute, San Antonio, TX. ^b Division of Comparative Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, OK.

By 2035, elderly individuals will outnumber children for the first time in US history resulting in substantial public health and financial impact. As we age, so does our im￾mune system, increasing our susceptibility to infections. Vaccinations are a vital tool to combat infections, yet they are much less effective in the elderly. Improving health outcomes in our aging population requires a better understanding of how the aging immune system impacts vaccine efficacy. To do this we are using the classical delayed-type hypersensitivity model of Mycobacterium bovis BCG vaccination and tuberculin skin test (TST). In this model, BCG vaccination generates BCG-specific immune cells that are recruited to the skin upon TST challenge. In our studies we BCG-vaccinate adult and elderly baboons and perform skin challenges eight weeks after vaccination by TST. Three days and one week after challenge we perform skin biopsies to examine BCG-specific cellular memory responses in the skin. We also determine BCG-specific memory responses in the blood to compare tissue and systemic responses within each vaccinated animal. We have shown that Mycobacterium tuberculosis (M.tb)-specific T cell responses are not altered in the blood of M.tb-infected aged individuals, suggesting that impaired immunity occurs in the tissue. Our data is supported by published literature showing elderly TST responses are decreased. We expect aged BCG-vaccinated baboons will have decreased and delayed vaccine-induced memory responses in the skin due to age-associated alterations, such as pro-inflammatory and oxidative factors, that hinder T cell migration to and function in the tissue.

Correspondence to: Julia M. Scordo, Texas Biomedical Research Institute, San Antonio, TX.
Email: jscordo@txbiomed.org

Transcriptomic analysis reveals upregulated microglia phagocytotic signals in the aging sub-ventricular zone

Ronald Cutler^a*, Erzsebet Kokovaya<sup>b

a</sup> Department of Cell Systems and Anatomy, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, United States. ^b The Barshop Institute on Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, United States.

The neural stem cell (NSC) pool in the subventricular zone (SVZ) experiences significant losses in number as well as proliferative capacity during aging, which functionally results in reduced cognition and neural plasticity. Microglia (MG) provide support for neurogenesis in the young SVZ while recent evidence from our lab indicates that during aging, MG attain an activated/pro-inflammatory phenotype that has been shown to contribute to agedependent losses in neurogenesis. However, the decline in the NSC pool remains to be fully elucidated and evidence of apoptosis to account for the loss of NSCs during aging is limited. We hypothesize that the aged SVZ MG phenotype may be indicative of increased phagocytic activity against NSCs and thus contributes to the decline of the NSC pool with aging. Here, we re-analyzed RNAsequencing data from multiple studies of the aging SVZ to test if the phagocytic transcriptional program is upregulated in aged MG or whether “eat-me” signals are upregulated in aged NSCs. We found significant upregulation of genes within the complement system (e.g. C1qa, C1qb, C3) in both aged NSCs and aged MGs, Trem2 receptor in aged MG, and scavenger receptor (e.g. Scarb1, Fcrls, Scarf2) in MG. Gene ontology (GO) analysis of upregulated genes revealed top enrichments in ‘innate immune response’ and ‘response to interferon-beta’ in aged NSCs and MGs where interferon-beta is known to activate phagocytic activity. However, almost all purinergic receptor related genes (e.g. P2ry12-13, P2rx7, P2ry2/6) were downregulated in aged MGs. Using network analysis, CXCL13 was identified to be highly upregulated during aging and at the intersection of the majority of GO processes. Soluble bridging molecules that are displayed on apoptotic cells such as CALR, Gas6, PROS1, and Tub were upregulated in aged NSCs. But, phosphatidylserine receptor expression, an anchored “eat-me” signal, was not detected in aged NSCs. Lastly, due to an enrichment of interferon signatures, transposon expression was examined which found over 500 differentially expressed transposons in aged NSCs and MGs. While phagocytosis signaling is complex and experimental validation is warranted, this transcriptomic study suggest that MG may indeed be more phagocytic in the aging SVZ.

Correspondence to: Ronald Cutler, Department of Cell Systems and Anatomy, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, United States.
Email: cutlerr@livemail.uthscsa.edu

A facile radiosynthesis and validation of novel tau PET tracer [F-18]-MK-6240

Ramesh Neelamegam ^a,b*, Daniel Yokell^b , Georges El Fakhri<sup>b

a</sup> Department of Radiology, University of Texas Health Science Center San Antonio, Research Imaging Institute, Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, San Antonio, Texas. ^b Gordon Center for Medical Imaging Massachusetts General Hospital, Harvard Medical School Boston MA United States.

[18F]MK-6240, a novel pyridine isoquinoline amine derivative developed by Merck, showed a high sensitivity and specificity for PHF-tau binding. In autoradiographic studies, MK-6240 showed high affinity to tangle-rich AD brain homogenates and large amounts of displaceable binding in the gray matter of AD brain sections. Absence of off-target binding of [18F]MK-6240 to MAO-A and MAO-B was confirmed in preclinical studies as well. Recent clinical studies have demonstrated that spatial patterns of MK-6240 binding were consistent with neuropathological staging of NFTs. Unlike flortaucipir ([18F] T807) and [18F]THK5351, off-target binding of [18F] MK-6240 was not observed in the basal ganglia and choroid plexus although mild tracer retention was observed in the substantia nigra and meninges. A novel tracer [18F] PI-2620 has also shown a lack of off target binding in the basal ganglia. Intriguingly, a preclinical analysis has suggested a high-affinity binding of [18F]PI-2620 to PSP and Pick’s disease brains. To confirm these initial observations, several clinical studies are ongoing in non-AD patients. [18F]MK-6240, a novel tau positron emission tomography (PET) tracer recently discovered for the in vivo detection of neurofibrillary tangles, has the potential to improve diagnostic accuracy in the detection of Alzheimer disease. Herein, we report the comparison of [18F]MK-6240 radiochemical yields with different F-18 elution reagents and different temperatures. Nucleophilic fluorination of the 5-diBoc-6-nitro precursor with potassium cryptand [18F]fluoride (K[18F]/K222) or tetraethylammonium [18F]fluoride (TEA[18F]) were performed by conventional heating, followed by thermal N-diBoc deprotection. Then radiochemical conversion (RCC) of the measured by radio-TLC. The crude product was purified by using ethanol and sodium acetate solution and further diluted with phosphate buffered saline then sterile filtered to obtain a solution suitable for injection. Results: HPLC analysis of formulated product revealed high radiochemical (>98%) and high chemical purity. Formulated [18F] MK-6240 maintained stability, as measured by radioHPLC and TLC, and met specifications for visual inspection and pH over a period of 6 hours. This work presents a novel automated radiosynthesis methods for [18F]MK- 6240 which eliminates the need for the presence of acid for deprotection of N-diBoc groups which makes the synthesis feasible on the Tracerlab FX2 N and other automated synthesis modules. It also eliminates the need for solid phase extraction (SPE) re-formulation of the purified product. The methodology described herein can facilitate multi-center trials and widespread use of this radiopharmaceutical for imaging tau protein in vivo.
Acknowledgements. We thank the Alzheimer’s Drug Discovery Foundation for generously providing funding for this research. We also thank David F. Lee Jr., Steven Gallo and Tim Beaudoin of the Massachusetts General Hospital for isotope production and technical support.

Correspondence to: Ramesh Neelamegam, Department of Radiology, University of Texas Health Science Center San Antonio, Research Imaging Institute, Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, San Antonio, Texas.
Email: neelamegam@uthscsa.edu

Optimization of C. elegans protein extraction method for proteasome kinetic assay and western blot analysis

Jessica L. Scheirer^a,b, Meghna N. Chinchankar^a,b, Alfred L. Fisher^c , Karl A. Rodriguez<sup>a,b

a</sup> Sam and Ann Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center San Antonio (UTHSCSA), San Antonio, TX. ^b Department of Cell Systems and Anatomy, UTHSCSA. ^c Division of Geriatrics, Gerontology, and Palliative Medicine, Department of Medicine, University of Nebraska Medical Center.

Accumulation of protein aggregates are a common pathology in many neurodegenerative disorders. Cellular maintenance of the proteome is overseen by a complex system of cellular pathways and molecular components. One of the ways cells remove protein aggregates is by utilizing the ubiquitin proteasome system (UPS). Concomitantly, proteasome activity declines with age leading to aggregation accumulation, which may be due to a function decline in the overall protein homeostasis network known to occur during the aging process. Consequently, investigating proteasome activity via kinetic based assays has become a regular method used in multiple laboratories. Caenorhabditis elegans is an animal model that has been used in varying fields of study such as neurobiology, genetics, developmental biology, aging and more recently biomedical and environmental toxicology, due to its wellcharacterized genome, short lifespan, large brood size, and 83% of its proteome is homologous to human genes. One drawback is C. elegans cuticle, an immensely elastic and durable exoskeleton, making protein extraction arduous and requiring large number of animals to achieve the necessary concentrations required for analysis. To overcome this obstacle, we have optimized a protein extraction method for C. elegans utilizing a small sample size for use in the kinetic proteasome assay and western blot analysis.

Correspondence to: Karl A. Rodriguez, Department of Cell Systems and Anatomy, UTHSCSA.
Email: RodriguezK@uthscsa.edu